This invention relates generally to liquid chromatography and more specifically relates to a solvent supply system for use in high performance column liquid chromatography.
Chromatography is a separation method wherein a mixture of components (called the "sample" or "sample mixture") is placed as a zone at one end of a system containing a stationary phase and a mobile phase. Each component of the sample distributes itself in dynamic equilibrium between the two phases in a ratio characteristic of that component. As a result, the flowing mobile phase causes each individual component zone to migrate at a characteristic rate, and the zones become separated after a period of time.
There are various types of chromatography, e.g., liquid chromatography, gas chromotography, thin-layer chromatography, etc. The major differences between these various chromatographic methods is the physical state of the mobile phase (gas or liquid), and the manner in which the stationary phase is supported, e.g., coated on an inert granular material packed in a tube, coated on an inner wall surface, etc. In each method, the separation mechanism is essentially the same, i.e., distribution of the sample components between a mobile phase and a stationery phase. When the method is used for chemical analysis, a detector is commonly placed at the far end of the system, so as to monitor the passage of the component zones as they emerge from the system. The signal from the detector is displayed on a recording device such as a strip chart recorder, and the record indicates both qualitative and quantitative information regarding the components of the sample.
It is often desirable for a chromatographic system to provide high resolution (a large degree of component separation with narrow zones), evenly spaced component zones, rapid separation, and a satisfactory record from a very small sample. The behavior of the system described in these terms may be called the "performance" of the system. It is well known in the chromatography art to improve system performance by changing one of the following system variables during the course of the analysis: temperature, chemical composition of the mobile phase, and flow rate of the mobile phase. For example, in gas chromatography the temperature of the system is often varied as a preselected function of time. This technique is known as "temperature programming", and it improves the performance of the system, especially with samples containing components which boil over a wide temperature range. Analagous to temperature programming in gas chromatography, is the use of "gradient elution" in liquid chromatography. Gradient elution refers to changing the chemical composition of the mobile phase (also called the "eluent" or "eluting solvent") as a function of time, thereby improving the performance of the system, especially with samples containing components which vary widely in chemical properties. The net effect of gradient elution is to shorten the retention time of compounds strongly retained on the columns without sacrifice in separation of early eluting compounds. Further details regarding the fundamentals of gradient elution techniques may be found in numerous sources in the prior art, as, for example, in the publication by L. R. Snyder appearing in Chromatography Review 7, 1 (1965).
A central concern pertinent to liquid chromatography apparatus of the type considered herein, is one of providing a proper flow of solvent to and through the chromatography column. Thus in the past, numerous and varied approaches have been utilized for supplying solvents to high performance liquid chromatography columns. A key requirement in this connection is one of providing a relatively non-pulsating, i.e. a constant flow of solvent -- in that the LC detector is sensitive to flow variations, and can provide erroneous readings and exhibit excessive noise in the presence of pulsing flow. Various approaches have been utilized in the past in order to enable such result, but in general, the prior art methodology directed at such end has involved highly expensive and overly complex mechanisms. Thus, in a typical example wherein a system is intended for operation in a gradient elution mode, i.e., by use of two distinct solvents, a dual pump arrangement may be utilized. Such arrangement requires two distinct pumps, including separate means for driving each of the pumps, which thus requires separate speed controls, etc.
In principle, it would seen that the cited problems arising in connection with the solvent pumping systems of the prior art, might be overcome by use of a single cylinder arrangement in cooperation with a relatively small displacement volume reciprocating piston. A principal deterrent to the use of this arrangement, however, has been the fact that the ensuing flow will, by its nature, be pulsating -- particularly at low flow rates. Further, the nature of the pulses present in the flow is such that they are not easily removed by filtering and the presence of such pulses can sharply limit the efficiency of the detector system. It should be understood in the foregoing connection that the word "piston" as used in this specification is intended to include both pistons where the seal remains fixed in relative position to the moving member and plungers where the seal is fixed with respect to the stationary cylinder.
It has in the foregoing connection, been long recognized that the aspect of the reciprocating pump which is principally responsible for an unacceptable pulsating flow is the fact that when the pump piston is driven by a simple crank shaft mechanism, the axial displacement of the piston as a function of time is sinusoidal. This implies the presence of equal time spaced pressure (or liquid pumping) pulses, alternating with fill periods of duration equal to the pressure pulse duration. In an effort to overcome this pattern, it has been proposed to drive the piston through suitably shaped cams. Pursuant to such approach these serve to alter the time displacement function of the pump piston so as to foreshorten the fill portion of the cycle in comparison to the pumping portion, and in some instances render the movement during pumping relatively linear in nature, i.e., the displacement is linear as a function of time. This sort of arrangement does have the advantage of changing the form of the pulsating pattern so as to diminish the pulsing and render filtering of the remaining pulses more feasible. However, the approach is less than satisfactory in a most important respect. In particular, the cam represents a fixed pattern, and thus provides a fixed relationship or ratio between the fill and pumping portions of the pump cycle. And yet, in many instances it is desired to have a capability for operation over various flow rates -- which indeed can vary very widely. If, however, the flow rate is increased by merely increasing the rate of cam rotation, then the fill portion of the cycle becomes successively shortened -- and can reach a point where insufficient feed time is available leading to cavitation and other problems.
In a copending application filed by the present inventors together with Pierre Achener on even date herewith, Ser. No. 630,103 and entitled HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SYSTEM, which application is assigned to the same assignee as is the present application, there is disclosed a liquid chromatography system which is particularly useful in overcoming the aforementioned flow problems. Said system includes a reservoir for a liquid mobile phase, a liquid chromatography column, reciprocating pumping means for pumping the mobile phase through the column, and motor means for driving the pumping means through successive reciprocation cycles. Means are provided further, for controlling the rotational speed of the motor throughout the reciprocation cycle of the pump so as to provide preselected average rotational speeds over predetermined subintervals of each successive reciprocation cycle. Application of the control cycle is synchronized with the pumping cycle so that the said speed control is properly applied over each successive reciprocation cycle.
A further problem evidenced in the prior art, including in the systems of the type just considered, is one of providing proper proportioning between the two solvents which are commonly utilized in the course of gradient elution work. The ratio of the solvents are typically changed as a function of time: and various approaches have in the past been utilized in order to achieve the desired ratios. Thus for example, a relatively complex approach may be employed wherein the solvents are fed from separate pumping means which are driven at differing speeds in accordance with the gradient setting.
It has also been known in the past to utilize proportioning valves for such purposes. Thus in one arrangement a single pump together with a pair of reservoirs and a holding coil are used. The proportioning valves are positioned at the high pressure side of the pump. The holding coil may first be filled with one solvent. The solvent from the second reservoir is pumped both to the first proportioning valve and to the holding coil; thereafter the first solvent is delivered from the coil to the second proportioning valve. These proportioning valves are alternately cycled to allow a prescribed quantity of each liquid to flow into a mixing chamber and thence into the liquid chromatography column.
In practice it has been found that schemes of the aforementioned type are not markedly effective in insuring accurate proportioning of the solvents. Because, further, the proportioning valves are on the high pressure side of the pump, the aforementioned holding coil is required. In addition, the high pressure utilized makes it necessary to employ a relatively costly construction for the proportioning valves -- or to sacrifice reliability if the costs are to be held within frugal limits.
In accordance with the foregoing, it may be regarded as an object of the present invention to provide high performance, high pressure chromatography apparatus, incorporating a relatively simple, relatively inexpensive reciprocating pump and additional elements which in cooperation with the pump provide highly nonpulsating uniform flow over a wide flow range; and wherein elements associated with the pump control enable simple and accurate control of solvent ratios when the apparatus is utilized with a plurality of solvents, i.e., in a gradient elution mode of operation.
It is a yet further object of the present invention, to provide a chromatography system of the foregoing high-pressure high performance type wherein the proportioning valves or similar elements utilized to provide a desired ratio between distinct solvents operate in a simple complementary fashion during a selected portion of the pump cycle, and function at the low pressure inlet side of the said pump.